Alveolar Macrophages Modulate Allergic Inflammation in a Murine Model of Asthma

EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 43, No. 5, 275-280, May 2011

Alveolar macrophages modulate allergic inflammation in a murine model of asthma

Bo-Ram Bang1,2, Eunyoung Chun1,2, of AMs in the context of airway inflammation should be Eun-Jin Shim1,2, Hyun-Seung Lee1,2, re-examined. Soo-Yeon Lee1,2, Sang-Heon Cho1,2, 1,2 1,2 Keywords: Kyung-Up Min , You-Young Kim 1,2,3 and Heung-Woo Park adoptive transfer; airway inflammation; alveolar macrophage; asthma; GM-CSF 1Department of Internal Medicine 2Institute of Allergy and Clinical Immunology College of Medicine Introduction Seoul National University Seoul 110-744, Korea Alveolar macrophages (AMs) are the most abun- 3Corresponding author: Tel, 82-2-2072-0699; dant cells in the alveolar spaces and conducting Fax, 82-2-742-2912; E-mail, [email protected] airways, and are known to be involved in immune DOI 10.3858/emm.2011.43.5.028 homeostasis in the respiratory tract (Wissinger et al., 2009). Asthma is a disease characterized by Accepted 18 March 2011 immune-mediated airway inflammation. Several Available Online 18 March 2011 reports have suggested that AMs suppress allergen- specific immune response and airway inflammation Abbreviations: AM, alveolar macrophage; BAL, bronchoalveolar (Thepen et al., 1991, 1992; Tang et al., 2001; Careau lavage; LPS, lipopolysaccharide; OVA, ovalbumin et al., 2010). However, this property seems to be dependent on the functional status of AMs. Sensi- tized AMs promote eosinophilic airway inflam- Abstract mation (Moon et al., 2007) and take part in acute asthma exacerbation by stimulating CD4-positive T The role of alveolar macrophages (AMs) in the patho- cells to secrete cytokines (Herbert et al., 2010). A genesis of asthma is still unknown. The aim of the pres- recent report has shown that allergen sensitization ent study was to investigate the effects of AM in the modulates AM function and only unsensitized AMs murine model of asthma. AMs were selectively de- protect against development of airway hyperrespon- pleted by liposomes containing clodronate just before siveness (Careau et al., 2006). allergen challenges, and changes in inflammatory In this study, we compared the effects of sensi- cells and cytokine concentrations in bronchoalveolar tized and unsensitized AMs on the reduction of lavage (BAL) fluid were measured. AMs were then allergic inflammation and cytokine secretion in a adoptively transferred to AM-depleted sensitized mice murine model of asthma. Phenotype changes were first evaluated after selective depletion of AM and changes were measured. Phenotypic changes in populations with liposomes containing clodronate AMs were evaluated after in vitro allergen stimulation. (dichloromethylenediphosphate or Cl2MDP). Then, AM-depletion after sensitization significantly in- sensitized or unsensitized AMs were adoptively creased the number of eosinophils and lymphocytes transferred into AM-depleted sensitized mice and and the concentrations of IL-4, IL-5 and GM-CSF in BAL numbers of inflammatory cells and cytokine con- fluid. These changes were significantly ameliorated centrations in bronchoalveolar (BAL) fluid were only by adoptive transfer of unsensitized AMs, not by measured. Finally, we evaluated the phenotypic sensitized AMs. In addition, in vitro allergen stim- changes in AMs after in vitro allergen stimulation. ulation of AMs resulted in their gaining the ability to produce inflammatory cytokines, such as IL-1β, IL-6 and TNF-α, and losing the ability to suppress GM-CSF Results concentrations in BAL fluid. These findings suggested that AMs worked probably through GM-CSF-depend- Changes in inflammatory cells and cytokine levels in ent mechanisms, although further confirmatory ex- BAL fluid after alveolar macrophage depletion periments are needed. Our results indicate that the role Administration of clodronate (Cl2MDP)-encapsulated 276 Exp. Mol. Med. Vol. 43(5), 275-280, 2011 liposomes resulted in significant increases in total circulation (Moon et al., 2007) after intranasal cell, eosinophil, and lymphocyte counts in BAL fluid challenge of OVA may explain the insignificant (Figure 1). Cytokine concentrations in BAL fluid, difference in the number of macrophages shown in IL-5 and GM-CSF, showed significant increases Figure 1. after AM-depletion (Figure 2). IFN-γ and IL-13 (data not shown) concentrations showed no differences and the levels of IL-12p70 were below the detec- Pro-inflammatory cytokine production from alveolar tion limit. macrophages after in vitro OVA stimulation AMs obtained from unsensitized mice expressed little or nopro-inflammatory cytokines (IL-1β, IL-6, Changes in inflammatory cells and cytokine levels in and TNF-α), but AMs obtained from sensitized BAL fluid after adoptive transfer of sensitized or mice produced significantly higher concentrations unsensitized alveolar macrophages to alveolar of pro-inflammatory cytokines after in vitro OVA macrophage depleted sensitized mice stimulation (Figure 3). When sensitized AMs were adoptively transferred to AM-depleted sensitized mice, there were no differences in BLA fluid inflammatory cell counts. Discussion However, adoptive transfer of unsensitized AMs significantly reduced total cell, eosinophil, and In the present study, we demonstrated that AM- lymphocyte counts in BAL fluid (Figure 1). Similarly, depletion after sensitization significantly increased adoptive transfer of sensitized AMs did not signifi- the number of eosinophilis and lymphocytes, and cantly decrease the concentrations of inflammatory the concentrations of IL-4, IL-5, and GM-CSF in cytokines in BAL fluid. However, adoptive transfer BAL fluid in a murine model of asthma. Moreover, of unsensitized AMs significantly reduced the con- increased numbers of eosinophilis and lympho- centrations of GM-CSF in BAL fluid (Figure 2). cytes and concentrations of GM-CSF in BAL fluid Macrophages recruited from regional tissue or after AM-depletion were significantly ameliorated

Figure 1. Changes in inflammatory cells in BAL fluid after alveolar macrophage depletion and after adoptive transfer of sensitized or unsensitized alveolar macrophage to alveolar macrophage depleted mice (A) total cell, (B) macrophages, (C) eosinophils, (D) neutrophils, (E) lymphocytes, LPS, lipopolysaccharide; sAM, sensitized alveolar macrophages; uAM, unsensitized alveolar macrophages; OVA, ovalbumin, *, P ＜ 0.05) Alveolar macrophage in asthma pathogenesis 277

Figure 2. Changes in inflammatory cytokine levels in BAL fluid after alveolar macrophage depletion and after adoptive transfer of sensitized or unsensitized alveolar macrophage to alveolar macrophage depleted mice (A, IL-4; B, IL-5; C, IFN-γ; D, GM-CSF; LPS, lipopolysaccharide; sAM, sensitized alveolar macrophages; uAM, unsensitized alveolar macrophages; OVA, ovalbumin; *, P ＜ 0.05) only by adoptive transfer of unsensitized AMs, not by sensitized AMs. The observations, in conjunc- tion with a previous report that unsensitized AMs can protect against development of airway hyperresponsiveness (Careau et al., 2006), suggest that AMs may have an important role in the development of asthma. In addition, we believe that the gain in ability to produce inflammatory cytokines, such as IL-1β, IL-6 and TNF-α, and the loss of ability to suppress GM-CSF concentrations in BAL fluid may contribute to the inability of sensitized AMs to play a protective role. Figure 3. Pro-inflammatory cytokine production from alveolar macrophages after in vitro ovalbumin stimulation (*, P ＜ 0.05). It was suggested that upon exposure to allergen, lung macrophages can convert to a Th1-oriented antigen presenting capacity by an IFN-γ-dependent mechanism, and that the participation of these changed significantly. This different observation cells in antigen presentation inhibits, to some may have been a result of differences in the mouse degree, the development of Th2 immune responses strain used and the experimental protocol, and in the bronchial mucosa (Tang et al., 2001). Along detailed mechanisms for these findings should be with previous reports, we found that populations of investigated further. To the best of our knowledge, eosinophils and lymphocytes in BAL fluid signifi- this is the first study showing that AMs may cantly increased after AM-depletion. However, we modulate allergic inflammation via GM-CSF in a murine model of asthma. could not observe any significant change in IFN-γ concentrations in BAL fluid after AM-depletion, A recent report showed that the transfer of whereas, the GM-CSF concentrations in BAL fluid unsensitized AMs 24 h prior to OVA challenge 278 Exp. Mol. Med. Vol. 43(5), 275-280, 2011 administered to AM-depleted, sensitized rats resulted but they lose this ability as they become sensitized in the abrogation of airway hyperresponsiveness, after repeated exposures to allergen. The whereas the transfer of sensitized AMs to AM- inflammatory phenotypic that we observed in the depleted, unsensitized rats did not significantly present study maybe one of the reasons for this alter airway responsiveness (Careau et al., 2006). finding. However, the authors did not observe any In summary, AM depletion significantly increased differences in the numbers of inflammatory cells in the numbers of total inflammatory cells, eosinophils BAL fluid after AM depletion and transfer and and lymphocytes in BAL fluid, and adoptive transfer concluded that the role of AMs in asthma is of unsensitized AMs effectively abrogated these unrelated to reduction in inflammatory cell recruit- change, probably through GM-CSF dependent ment, but instead may involve other mechanisms mechanisms. Our results indicate the role of AMs modulating airway hyperresponsiveness. The ob- in the context of airway inflammation should be servations are contrary to our findings. However, re-examined. airway hyperresponsiveness generally correlates reasonably well with airway inflammation (Busse and Lemanske, 2001). AMs have long been reco- Methods gnized to be activated in asthmatic inflammation, with evidence of increased secretion of cytokines Mice such as tumor necrosis factor α and IL-6 (Gosset Female C57BL/6 mice (6-week old) were purchased from et al., 1991). In addition, it has been revealed that the Jackson Laboratory (Bar Harbor, ME). All animals were AMs from atopic asthmatic subjects, but not from maintained in filter-top cages under specific pathogen-free atopic non-asthmatic subjects, enhance IL-4 and conditions at the animal facility of the Seoul National IL-5 production by CD4+ T cells upon stimulation University College of Medicine (Seoul, Korea). Experi- with allergen (Tang et al., 1998a, 1998b). Recently, it mental procedures were performed with the approval of the was reported that the airway epithelial cell derived Seoul National University Institutional Animal Care and IL-33 amplified an alternative activation and che- Use Committee (IACUC) in accordance with the guidelines of IACUC. mokine productions from macrophages (Kurowska- Stolarska et al., 2009). Taken together, it is clear that AM plays a potential role in the modulation of Experimental protocols for a murine model of asthma local inflammation. and alveolar macrophage depletion However, in the present study using a murine For sensitization, mice were anesthetized and ovalbumin model of asthma, we found that airway inflam- (OVA, Sigma Chemical Co., St. Louis, US) plus 0.1 μg of mation was not inhibited and even was increased lipopolysaccharide (E. coli O55:B5, Calbiochem, San after AM depletion. Similar findings were observed, Diego, US) in 30 μl of phosphate buffered saline (PBS) were in a rat model of trimellitic anhydride-induced occu- instilled intranasally on days 1, 2, 3, and 8. On days 7 and pational asthma (Valstar et al., 2006). The authors 8 after the last sensitization with OVA plus LPS (days 15, reported that AM depletion augmented the trime- 16), mice were intranasally challenged with OVA only llitic anhydride-induced tissue damage and inflam- (Supplemental Data Figure S1A). For AM depletion, mice mation 24 h after challenge. The reason for this were first challenged with OVA plus LPS as described phenomenon could be based on the functional previously (Elder et al., 2004), and then received 30 μl of clodronate (Cl2MDP)-encapsulated liposomes (Roche status of the AMs. As mentioned previously, AMs Diagnostics GmbH, Mannheim, Germany) on 3 conse- play a key role in the maintenance of immuno- cutive days (days 12-14) just before the first OVA challenge logical homeostasis in the respiratory tract. As (Supplemental Data Figure S1B). Confirmation that more examples, allergen challenge of AM-depleted sensi- than 75% of alveolar macrophages were depleted without tized rats results in a highly elevated immuno- affecting other cell types were performed by counting the globulin E concentrations, large influxes of T and B macrophages in BAL fluid from treated and untreated mice. cells into the lungs, and increase in airway hyperresponsiveness (Thepen et al., 1991, 1992; Careau Isolation of alveolar macrophages and adoptive et al., 2006). We also observed that inflammatory transfer cells in BAL fluid significantly increased when AMs were depleted in mice in a murine model of Sensitized AMs were obtained from lungs of sacrificed asthma, and these increases were attenuated by mice 7 days after the last sensitization (day 15 in Supple- the adoptive transfer of AMs, although significant mental Data Figure S1A), and unsensitized AMs were obtained from lungs of sacrificed mice who did not undergo changes were found only when unsensitized AMs sensitization. The lungs were perfused via the right were transferred. This suggest that unsensitized ventricle with 5 ml of PBS and excised and minced lung AMs are capable of reducing allergic inflammation, tissue was incubated for 15 min at 37oC in 0.5% EDTA. To Alveolar macrophage in asthma pathogenesis 279 obtain a single cell, the minced tissue was grinded on the TNF-α in cell culture supernatant were measured using the strainer with complete RPMI (cRPMI) that was supplemented Bio-Plex cytokine assay. 10% FBS, L-glutamine, sodium pyruvate, penicillin-strep- tomycin, and erythrocytes were lysed by treatment with ammonium chloride solution (StemCell technologies, Vancouver, Statistical analysis Canada). The cells were washed twice with RPMI, and All data are reported as the mean ± standard error (SE) of centrifuged at 1500 rpm for 10 min. We defined AMs as - + + the means. A two-tailed Student t-test was used for CD11b CD11c cells. To isolate CD11c cells, auto MACS analysis and differences were considered significant for P sorter (Miltenyi Biotec, Auburn, CA) with anti-CD11c anti- ＜ 0.05. body conjugated to magnetic beads were used in accordance with manufacturer’s instructions. Isolated CD11c+ - cells were then stained with anti-CD11b to sort CD11b Supplemental data CD11c+ cells. The stained cells were gated into CD11b- CD11c+ cells according to expressions of CD11b or CD11c Supplemental data include a figure and can be found with molecule and sorted using the live sterile cell sorting this article online at http://e-emm.or.kr/article/article_files/ system (FACSAria system, Becton Dickinson, San Jose, SP-43-5-02.pdf. CA). The purity of CD11b- CD11c+ cells was ＞ 97%. Isolated sensitized or unsensitized AMs (1×105 cells) were transferred intratracheally to AM-depleted sensitized Acknowledgements mice on the day of the first OVA challenge (day 15, This work was supported by a grant of the Korea Health- Supplemental Data Figure S1C). Mice were sacrificed on care technology R&D Project, from Ministry of Health and day 18, and lung inflammation was evaluated (Supple- Welfare, Republic of Korea (A102065), and by a grant from mental Data Figure S1C). Seoul National University Hospital (0420070550).

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